PROJECT SUMMARY The development of highly selective anti-tumor agents continues to be a challenge in drug discovery and this fact has motivated researchers to search for new molecular targets that allow for the discovery of more selective anticancer agents. Recent collaborative work from the Webb laboratory and others has shown that small molecules that interact with certain spliceosome proteins (such as the SF3B1 protein) target a key vulnerability in certain cancers. Progress in this area has been facilitated by the development of potent small molecules (such as sudemycin D6) that we have developed, which show potent in vivo antitumor activity including complete tumor regression in animal models without significant toxicity. Additionally we have contributed to an improved understanding of the mechanism of action of these molecules with our early observation that these small molecules have potent effects on alternate splicing and this activity is the hallmark feature of these antitumor compounds. This validation of the spliceosome as a therapeutic target for cancer presents an opportunity for the discovery of a family of new therapeutic targets and new agents as probes for understanding the basic cell biology of splicing and, potentially, as starting points for new anticancer agents. This grant application proposes the use of a new triple-exon skipping assay that has very recently shown its utility in the discovery new active molecules as the starting points in the development of potent drug-like in vivo probes, which modulate the processing of pre-mRNA. We propose the screening of a larger and more diverse compound library using our novel screen along with the validation of the new active molecules by the implementation of a series of biological assays. We believe that this novel assay will uncover new active molecules that will be used to developing a better understanding of the process of pre-mRNA splicing and its role in diseases such as cancer. Our path forward involves scaling up the screen and implementing a series of biochemical and cell based assays that have already been implemented on a pilot scale. The confirmatory assays would include biochemical assays to determine inhibition against the cdc-like kinase family (CLK 1-4), DYRK1A, and SRPK and cell-based assays to show binding to SF3B1. This project will focus entirely on novel small molecules that target the spliceosome, which is an important emerging target for cancer therapy. The goal for this stage of the project is to identify and validate probe molecules for known targets and importantly to also identify compounds that modulate splicing by targeting previously unidentified splicing targets. A goal for future grant applications would be the development of structure-activity relationships for new compounds and the full characterization of newly identified spliceosome targets.